ABSTRACT Astrocytes are important in AD pathogenesis by two major mechanisms. The first mechanism involves interactions with neurons, microglia, and oligodendrocytes and the second is by secretion of significant quantities of A? to increase amyloid burden in the brain. Both AD and brain cancer increase with age, however, several comprehensive longitudinal studies with more than one million participants have shown an inverse relationship between the two diseases; the risk of AD in individuals with cancer was decreased by 35% [1-7]. Our previous work analyzed microarray datasets of AD (n=524) and glioblastoma (GBM) (n=1091) cohorts and investigated the transcriptional signaling mediating the inverse relationship between the two diseases [8]. We found that AD and brain cancer have common and distinct deregulated transcriptional signals. In this supplemental study, we will focus on how tumor in the brain reduces AD pathogenesis. Our original R01 investigates astrocyte-tumor interactions in brain metastatic tumor. We developed novel experimental and computational approaches to study astrocyte?tumor paracrine and astrocyte?astrocyte autocrine signals for potential therapeutic strategies for brain metastatic tumors. In AD, astrocytes are under astrocyte?neuron paracrine and astrocyte?astrocyte autocrine regulations, which are critical in causing A? over-production from both neurons and astrocytes. We will expand the R01 study to explore whether the transcriptional changes in astrocytes upon interacting with cancer cells in the brain would offer new insights to reduce the vicious astrocyte?neuron or astrocyte?astrocyte interactions and reduce amyloid burden in AD. Our central hypothesis is that the astrocyte-tumor interactions would reduce A? production from neurons and astrocytes, facilitate A? break-down by astrocytes, or both. To test this hypothesis, we set forth the following specific aims: 1) generate single-cell RNAseq data of astrocytes, neurons, and tumor cells from the brain of brain metastatic model in aged mice and 2) perform astrocyte-tumor-neuron crosstalk analysis using the CCCExplorer platform as in our original R01. These two aims can be finished in one year. We expect to identify astrocyte-centric transcriptional signals that down-regulate A? production in neurons and/or astrocytes, and up-regulate the proteasomal and phagocytic breakdown of A?. After the supplement study, we will further explore: 1) the post-transcriptional change of the identified signaling molecules, including secretable ligands, membrane receptors, and intra-cellular signaling proteins by ELISA, western blot, and immunostaining; 2) the function of the key signaling molecules in in vitro Alzheimer?s-in-a-dish models [9, 10] as in Wong?s current NIA R01 project on Alzheimer?s disease; and 3) the therapeutic potential of the astrocyte signaling using AD transgenic mouse models.